Dental caries is one of the most common bacterial infections in humans and remains untreated in many underdeveloped countries. Based on biochemical, epidemiological and animal experiments, Streptococcus mutans is considered to be the principal etiological agent of dental caries. The ability to metabolize carbohydrates and to adhere to and form tenacious biofilms on the tooth surfaces are believed to be critically associated with the cariogenicity of this human pathogen. S. mutans synthesizes glucans from sucrose by three glucosyltransferases (Gtf), and adheres firmly to tooth surfaces with their cooperation. It also produces glucan-binding proteins (Gbps) which play major roles in virulence. The specific mechanisms governing regulation of exopolysaccharide synthesis in S. mutans have yet to be discovered. However, recently it was found that an orphan response regulator, GcrR, modulates the expression of at least one Gtf and one Gbp gene. Remarkably, inactivation of gcrR drastically reduces biofilm formation and cariogenesis in rat. Therefore, GcrR appears to be very important for pathogenesis of this organism. In addition, GcrR shows extensive sequence homology (>80%) with the pathogenic group A streptococcus (GAS) CovR, a response regulator that controls as much as 15% of the GAS genes including many important virulence factors. Given the high degree of similarity with CovR and its effect in biofilm and cariogenesis, one would expect that GcrR may be a global regulator of S. mutans. Therefore, we have chosen to focus on gene regulation by GcrR with the following Specific Aims. In Aim 1, we will identify the gcrR regulon by DNA microarray and in vitro DNA binding assays using purified GcrR protein. We will confirm our results with proteomics approach. In Aim 2, we will determine the mechanisms of gene regulation by GcrR. We will identify and characterize the GcrR binding motif(s) on the promoters of genes (such as gtfD, gbpC) regulated by GcrR. In Aim 3, we will study the regulation of gcrR expression. Since the cognate sensor kinase of GcrR is absent in the nearby gcrR locus, using both biochemical and genetic approaches we will identify the cognate sensor kinase to understand gcrR regulation. This (investigationwill promote our understanding of molecular mechanisms of gene regulation and signal transduction in S. mutans and facilitate the development of therapeutic approaches aimed at controlling formation of plaque biofilm and subsequent cariogenesis.